Browsing by Subject "Fluorescent microscopy"

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    Cell free expression in emulsions and vesicles.
    (2010-08) Monina, Nadezda
    In this study we address the effects that membrane composition has on expression within two common forms of encapsulation: the single layered micelle and the bilayered vesicle. We then proceed to show the effects of oxygen on transcription machinery and the role the membrane plays in optimizing oxygen concentration for protein expression. In each case, the surfactant encapsulation is prepared in mineral oil and encloses an Escherichia coli cytoplasmic extract. Measurement of expression efficiency is conducted by both endpoint and kinetic measurement of eGFP (enhanced green fluorescent protein) concentrations via fluorescent microscopy. In the case of micelle encapsulation, results show that micelles of long block copolymer (LBCP) have the highest rate of expression as well as final concentration at 8 hours. Results also show a large discrepancy in eGFP expression between well oxygenated and less oxygenated environments. Similarly, a comparison between lipids suggests longer fatty acid tail length corresponds to more effective inhibition of oxygen diffusion leading to greater expression efficiency. Further expression tests in vesicles show membranes composed of phosphotidylcholine (PC) lipid mixed with LBCP have the most efficient eGFP as well as α hemolysin expression among vesicles of differing composition. With this information comes a newfound knowledge of the effects of synthetic membrane composition, oxygen concentration and the relationship between these two parameters on protein expression.